Jonathan Tucci

Obesity is associated with residual leukemia following induction therapy for childhood B- precursor acute lymphoblastic leukemia

Obesity is associated with poorer event-free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). Persistent minimal residual disease (MRD) in the bone marrow as measured by multidimensional flow cytometry (MDF) is a key early prognostic indicator and is strongly associated with EFS. We therefore hypothesized that obesity during induction would be associated with positive end-of-induction MRD (≥0.01%). We analyzed MDF of end-induction bone marrow samples from a historical cohort of 198 children newly diagnosed with B-precursor ALL (BP-ALL) and treated with Childrens Oncology Group induction regimens. We assessed the influence of body mass index on risk for positive end-induction MRD in the bone marrow. In our cohort of BP-ALL, 30 children (15.2%) were overweight and 41 (20.7%) were obese at diagnosis. Independent of established predictors of treatment response, obesity during induction was associated with significantly greater risk for persistent MRD (odds ratio, 2.57; 95% confidence interval, 1.19 to 5.54; P = .016). Obesity and overweight were associated with poorer EFS irrespective of end-induction MRD (P = .012). Obese children with newly diagnosed BP-ALL are at increased risk for positive end-induction MRD and poorer EFS.

Adipocytes cause leukemia cell resistance to daunorubicin via oxidative stress response

Adipocytes promote cancer progression and impair treatment, and have been shown to protect acute lymphoblastic leukemia (ALL) cells from chemotherapies. Here we investigate whether this protection is mediated by changes in oxidative stress. Co-culture experiments showed that adipocytes protect ALL cells from oxidative stress induced by drugs or irradiation. We demonstrated that ALL cells induce intracellular ROS and an oxidative stress response in adipocytes. This adipocyte oxidative stress response leads to the secretion of soluble factors which protect ALL cells from daunorubicin (DNR). Collectively, our investigation shows that ALL cells elicit an oxidative stress response in adipocytes, leading to adipocyte protection of ALL cells against DNR.

Abstract 5: Using stable isotope lipidomics to identify adipocyte-induced alterations in the acute lymphoblastic leukemia lipidome

Obesity is associated with the development and progression of many cancers, including acute lymphoblastic leukemia (ALL). We have shown that ALL cells induce adipocyte lipolysis and take up adipocyte-derived free-fatty acids (FFAs). Here, we use targeted lipidomics with carbon-13 labeling to 1) determine which adipocyte-derived FFAs are taken up by ALL cells, and 2) identify the specific alterations in the ALL cell lipidomic profile caused by co-culture with adipocytes. Mouse pre-adipocytes (3T3-L1) were differentiated into adipocytes in the presence of U13C-glucose to allow 13C incorporation into adipocyte lipids. ALL cells were then cultured alone or co-cultured over 13C-labeled or non-labeled adipocytes for 72 hours. Following co-culture, ALL cells were harvested and analyzed by nanospray desorption electrospray ionization mass spectrometry (nanoDESI-MS). Lipidomic spectra were analyzed to characterize uptake of adipocyte-derived FFAs identifying 13C-enrichment in ALL lipid moieties. The relative intensities of selected lipid peaks were compared between conditions, after normalization, to evaluate the effects of the presence of adipocytes on ALL lipidome. Adipocytes differentiated in the presence of U13C-glucose incorporated 13C into numerous triglyceride moieties. In ALL cells co-cultured with labeled adipocytes, 13C enrichment was identified in FFA and phospholipids. Labeling of oleic acid primarily occurred in groups of two, suggesting incorporation of U13C-glucose into adipocyte FFAs through acetyl-CoA-mediated lipogenesis. Similarly, 13C enrichment of ALL cell phospholipids occurred in groups of two and three, mirroring adipocyte incorporation of U13C-glucose into phospholipids through both acetyl-CoA and glycerol. Moreover, ALL cell unsaturated FFAs had a greater 13C enrichment than saturated FFAs (Unsaturated FFAs: 14.1±5.5 vs. Saturated FFAs: 6.6±2.3, p = 0.02; n = 5). When co-cultured over adipocytes, ALL cells contained significantly more FFAs than ALL cells alone (Oleic Acid: 63.0±49.2 vs. 24.1±7.4, p = 0.02; Stearic acid: 20.9±17.5 vs. 8.3±2.2, p = 0.03; Palmitoleic Acid: 11.5±9.8 vs. 3.3±1.0, p = 0.01; Palmitic Acid: 15.1±11.7 vs. 5.5±1.4, p = 0.01; n = 5). Using stable-isotope lipidomics, we can effectively identify and quantify which ALL cell lipids are derived from nearby adipocytes. This methodology provides comprehensive insight into the cancer cell lipidome and can be extended to understand how genetics, the microenvironment, and treatment affect cancer lipid metabolism. Citation Format: Jonathan Tucci, Katy Margulis, Cheng-Chih Hsu, Wesley Dixon, Richard N. Zare, Steven D. Mittelman. Using stable isotope lipidomics to identify adipocyte-induced alterations in the acute lymphoblastic leukemia lipidome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5.

Adipocytes Sequester and Metabolize the Chemotherapeutic Daunorubicin

Obesity is associated with poorer outcome for many cancers. Previously, we observed that adipocytes protect acute lymphoblastic leukemia (ALL) cells from the anthracycline, daunorubicin. In this study, it is determined whether adipocytes clear daunorubicin from the tumor microenvironment (TME). Intracellular daunorubicin concentrations were evaluated using fluorescence. Daunorubicin and its largely inactive metabolite, daunorubicinol, were analytically measured in media, cells, and tissues using liquid chromatography/mass spectrometry (LC/MS). Expression of daunorubicin-metabolizing enzymes, aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3), in human adipose tissue, were queried using public databases and directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Adipocytes absorbed and efficiently metabolized daunorubicin to daunorubicinol, reducing its antileukemia effect in the local microenvironment. Murine studies confirmed adipose tissue conversion of daunorubicin to daunorubicinol in vivo. Adipocytes expressed high levels of AKR and CBR isoenzymes that deactivate anthracyclines. Indeed, adipocyte protein levels of AKR1C1, AKR1C2, and AKR1C3 are higher than all other human noncancerous cell types. To our knowledge, this is the first demonstration that adipocytes metabolize and inactivate a therapeutic drug. Adipocyte-mediated daunorubicin metabolism reduces active drug concentration in the TME. These results could be clinically important for adipocyte-rich cancer microenvironments such as omentum, breast, and marrow. As AKR and CBR enzymes metabolize several drugs, and can be expressed at higher levels in obese individuals, this proof-of-principle finding has important implications across many diseases. Implications: Adipocyte absorption and metabolism of chemotherapies can reduce cytotoxicity in cancer microenvironments, potentially contributing to poorer survival outcomes. Mol Cancer Res; 15(12); 1704–13. ©2017 AACR.

Adipocyte differentiation is affected by media height above the cell layer

Background:3T3-L1 cells have been widely used as a model for adipogenesis. However, despite its popularity, differentiation of this cell line has been reported to be inconsistent with low efficiency.Objective:To investigate the effect of media height during adipocyte differentiation on lipid accumulation and adipokine secretion in mature adipocytes.Methods:Three cell lines (3T3-L1, OP9 and ChubS7) were used to test the influence of media volume on adipogenesis. Total lipid content and lipid droplet size and number were quantified. Adipocyte related gene expressions were quantified during the course of differentiation. Secretion of leptin and adiponectin from mature adipocytes were measured using enzyme-linked immunosorbent assays. The influence of oxygen partial pressure on adipogenesis was investigated using three oxygen percentages: 5, 21 and 30%. Insulin sensitivity was measured by insulin inhibition of isoproterenol-induced lipolysis and phosphorylation of insulin receptor substrate-1.Results:A lower media height during adipogenesis increased total lipid accumulation, NEFA release and leptin and adiponectin secretion in mature adipocytes. Insulin sensitivity was not affected by media height during differentiation.Conclusion:Media height during adipogenesis was inversely correlated with lipid content in mature adipocytes. To achieve a high lipid content and greater adipokine secretion, it is best to use a low media volume during differentiation.

Salsalate treatment improves glycemia without altering adipose tissue in nondiabetic obese hispanics

Salsalate treatment has well‐known effects on improving glycemia, and the objective of this study was to examine whether the mechanism of this effect was related to changes in adipose tissue.

A novel biopsy method to increase yield of subcutaneous abdominal adipose tissue

Collection of abdominal subcutaneous adipose tissue (SAT) for research testing is traditionally performed using punch biopsy or needle aspiration techniques, yielding small amounts of very superficial SAT (100–500 mg). Although liposuction techniques can be used to obtain large amounts of SAT, these approaches can compromise the integrity of the adipose tissue. Therefore, we investigated a novel method using a 6-mm Bergström side-cutting biopsy needle to acquire suitable amounts of intact abdominal SAT for multiple complex studies such as flow cytometry, RNA extraction, ex vivo expression of molecular and post-translational protein mediators, and histology. Fifty biopsies were obtained from 29 participants using a Bergström biopsy needle, applying transient manual suction and shearing large pieces of fat within the inner-cutting trochar. Eighteen of the biopsies were performed under ultrasound guidance, whereby we successfully sampled deep SAT (dSAT) from below Scarpa’s fascia. The average weight of SAT sampled was 1.5±0.4 g. There was no clinically important bleeding or ecchymosis on the abdominal wall and no infection occurred with this procedure. The 6-mm Bergström biopsy needle yielded substantially more SAT than what has been obtained from superficial procedures and, for the first time, allowed sampling of dSAT by a percutaneous approach.

Abstract 4339: Acute lymphoblastic leukemia cells stimulate adipocyte lipolysis and utilize adipocyte-derived free-fatty acids for proliferation

Obesity promotes the pathogenesis of many cancers, including acute lymphoblastic leukemia (ALL), the most common childhood malignancy. We have shown that ALL cells interact with adipocytes in vivo and in vitro, and that adipocytes protect ALL cells from chemotherapies. Since cancer cells require free fatty acids (FFA) for energy and as molecular building blocks, we hypothesize that ALL cells gain a survival advantage by stimulating adipocyte lipolysis and using adipocyte-derived FFAs. To test whether ALL cells stimulate adipocyte lipolysis, 3T3-L1 adipocytes were exposed to ALL-conditioned media for 72 hours, and glycerol release and adipocyte lipid content were measured. To determine whether ALL cells take up adipocyte-derived FFA, adipocytes were pre-labeled with fluorescent FFA (BODIPY), and then co-cultured with murine (8093) and human (BV173, RS4;11, Molt4, Nalm6, SEM, SupB15) ALL cells in a Transwell system. ALL uptake of BODIPY-FFAs was analyzed via flow cytometry. To determine the fate of FFA in ALL cells, ALL cells were incubated with BODIPY-FFA and evaluated by confocal and live cell microscopy, and lipid classes resolved through thin layer chromatography (TLC). Real-time PCR was employed to analyze adipocyte-induced alterations in the transcription of key enzymes in the ALL de novo lipogenesis pathway. Finally, TOFA, an acetyl-CoA carboxylase 1 (ACC1) and steroyl-CoA desaturase 1 (SCD1) inhibitor, was used to block de novo lipogenesis in ALL cells in vitro, and evaluate the ability of ALL cells to use adipocyte-derived FFAs for proliferation. ALL-conditioned media stimulated 3T3-L1 lipolysis (191.0 vs. 165.5 mg/L glycerol, n=1), reducing adipocyte lipid content as quantified with Oil Red O by 16.8 ± 7.1% (p = 0.047). When murine or human ALL cells were co-cultured with BODIPY-labeled adipocytes, they accumulated the fluorescent label within 48 hours (97-98% of cells labeled positive). TLC, live cell imaging and confocal microscopy demonstrated the majority of this label accumulated within ALL cell phospholipid membranes and triglyceride-laden lipid droplets. Adipocyte co-culture reduced expression of lipogenic enzymes such as fatty acid synthase (FAS), ACC1 and SCD1 in ALL cells. Finally, while TOFA inhibited ALL cell proliferation (6.1 ± 1.1x104 vs. 41.9 ± 4.7x104 cells, p=0.004, n=3), this proliferation was partially rescued by adipocyte conditioned media (ACM, 29.8 ± 9.2 x104, p=0.045 vs. TOFA alone, n=3). Therefore, we have demonstrated that ALL cells stimulate adipocyte lipolysis and use adipocyte-derived FFAs to supplement de novo lipogenesis and proliferation. This may contribute to the effect of obesity on ALL development and relapse. Citation Format: Jonathan Tucci, Xia Sheng, Steven D. Mittelman. Acute lymphoblastic leukemia cells stimulate adipocyte lipolysis and utilize adipocyte-derived free-fatty acids for proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4339. doi:10.1158/1538-7445.AM2014-4339

Switch to low-fat diet improves outcome of acute lymphoblastic leukemia in obese mice

BackgroundIt is becoming increasingly recognized that weight and nutritional status can impact cancer survival. We have previously shown that obese mice with syngeneic acute lymphoblastic leukemia (ALL) have poorer response to chemotherapy treatment than control mice. We therefore investigated whether dietary intervention could improve outcome from the most common pediatric cancer, ALL.MethodsDiet-induced obese (DIO) mice raised on a 60% calories from fat diet and control mice were implanted with syngeneic ALL cells. Some DIO mice were switched to the low-fat control diet. Survival from ALL was assessed without or with chemotherapy treatment starting at the time of the diet switch. Cells from DIO mice before and after diet switch were assessed by FACS for BrdU incorporation and phosphorylation status of AKT, S6K, and EIF2a. Similar experiments were done with human ALL xenografts. Mouse and human ALL cells were cultured in media with 10% or 5% fetal bovine serum, and sensitivity to chemotherapies assessed.ResultsDIO mice had poorer survival (17%) after vincristine monotherapy than control mice on a 10% low fat diet (42%; n = 12/group; p = 0.09, log rank). However, switching obese mice to the low-fat diet prior to initiation of vincristine led to dramatically improved survival (92%, p < 0.01 vs both other groups). In vitro, FBS restriction made murine and human ALL cells more sensitive to vincristine. Interestingly, while serum restriction enhanced ALL sensitivity to dexamethasone and l-asparaginase, dietary switch did not improve survival of DIO mice treated with either drug in monotherapy. Thus, it appears that dietary intervention has a unique effect to improve ALL cell sensitivity to vincristine in vivo.ConclusionsWe report herein that a dietary intervention can improve ALL outcome in a preclinical model. Further work is needed to identify the mechanisms of this effect and investigate potential impact on human leukemia in patients.

Mouse Models to Study Obesity Effects on Hematologic Malignancies

In the present chapter, we discuss how mouse models have been used to investigate the interactions between obesity and hematologic malignancies. We begin with a brief overview of mouse models of obesity, followed by a description of how hematologic malignancies have been studied in these models. Unfortunately, the study of obesity and hematologic malignancies in mice is not as advanced as in other cancers such as breast and colorectal carcinoma. Therefore, where no studies exist, we describe how current models of hematologic malignancies could be made obese so that the effects of obesity on these malignancies could be studied.